Evaluation of Prophage Gene Revealed Population Variation of ‘Candidatus Liberibacter Asiaticus’: Bacterial Pathogen of Citrus Huanglongbing (HLB) in Northern Thailand

‘Candidatus Liberibacter asiaticus’ is a non-culturable bacterial pathogen, the causal agent of Huanglongbing (HLB, yellow shoot disease, also known as citrus greening disease), a highly destructive disease of citrus (Rutaceae). The pathogen is transmitted by the Asian citrus psyllid: Diaphorina citri Kuwayama. Recent studies, have shown that the HLB pathogen has two prophages, SC1 that has a lytic cycle and SC2 associated with bacterial virulence. This study aimed to search for SC1 and SC2 prophages of HLB in mandarin orange, sweet orange, bitter orange, kumquat, key lime, citron, caviar lime, kar lime, pomelo and orange jasmine from ve provinces in Northern Thailand. A total of 216 samples collected from Northern Thailand during 2019 and 2020 were studied. The results revealed that 62.04% (134/216) citrus samples were infected with the ‘Ca. L. asiaticus’ the bacterial pathogen associated with citrus HLB. The prophage particles are important genetic elements of bacterial genomes that are involved in lateral gene transfer, pathogenicity, environmental adaptation and interstrain genetic variability. Prophage particles were evaluated in the terminase gene of SC1 and SC2-type prophages. The results of the analysis of the prophage terminase genes of SC1 and SC2 revealed four groups of samples in Northern Thailand.The rst group was the population of ‘Ca. L. asiaticus’ which was non-prophage; the frequency was 7.46% (10/134) from the total infected citrus samples. The second and third groups contained one prophage sequence with 1.49% (2/134) containing SC1-type prophage and 64.93% (87/134) containing SC2-type prophage. The fourth group contained both prophage sequence with 26.12% (35/134) containing SC1 and SC2-type prophages.Samples of leaves showing various HLB symptoms were collected from infected citrus trees which correlated with the prophages. In the rst group (non-prophage) the symptoms were much less severe than other groups which had prophages. On the other hand, the third group containing the SC2-type prophage and fourth group containing SC1 and SC2-type prophages showed the most severe symptoms. The evaluation of the prophage effect on HLB-induced leaf symptoms showed that isolates with the SC2-type prophage particles produced the most severe symptoms.


Introduction
Citrus is one of the most popular fruit and is a major fruit crops grown worldwide (Li et al. 2010; Matheyambath et al. 2016). Belonging to the Rutaceae family, the crop is most extensively cultivated in tropical and sub-tropical regions.The genus Citrus consists of approximately 1300 species and some hybrids evolved by crossing between native varieties such as Citrus reticulata (mandarin), C. sinensis (orange), C. aurantifolia (lime), C. limon (lemon), and C. maxima (pomelo), etc. (Berk 2016). According to the FAO 2017, the annual production of citrus fruits have recorded a brisk growth in the past decades, rising from approximately 30 million tons in 1960 to over 147 million tons in 2017, with C. sinensis representing approximately 50% of global citrus production, C. reticulata (22%), C. limon/C. aurantifolia (11%) and C. paradisi (6%). However, Citrus spp. has recently been hampered by environmental and disease pressures (Wang et al. 2017).
Huanglongbing (HLB), also known as citrus greening, is the most destructive disease of citrus worldwide.
The pathogen is non-culturable, phloem-limited and associated with three species of α-Proteobacteria: 'Candidatus Liberibacter asiaticus' (Las), 'Ca. L. africanus' (Laf) and 'Ca. L. americanus' (Lam) (Bové 2006). The pathogens are transmitted by psyllids including the Asian citrus psyllid: Diaphorina citri Kuwayama, Cacopsylla (Psylla) citrisuga (Hemiptera: Psyllidae) and African citrus psyllid: Trioza erytreae (DelGuercio) (Cen et al. 2012;Gottwald 2007). Trioza erytreae was found in Africa (Aubert 1987) and Diaphorina citri was found in Asia, North America and South America (Bové J 2006). In Thailand, citrus huanglongbing is the main threat to the citrus industry since this disease rst appeared in Thailand in the 1960's. The HLB epidemics caused a signi cant decline in citrus fruit yield. (Roistacher 1996;Schwarz et al. 1973a;Schwarz et al. 1973b) Bacteriophages are the most abundant organisms in the biosphere with an estimated 10 31 particles (Wommack and Colwell 2000). Bacteriophages can multiply in living bacterial cells using bacterial materials and phage enzymes by two mechanisms, rstly, the lytic cycle as phage and secondly, the lysogenic cycle as prophage. Phages or prophages have long been used to distinguish bacterial isolates from different environmental and clinical sources (Grange 1975;Hatfull 2010;Salloum et al. 2011).
Prophages are important genetic elements of bacterial genomes which are involved in lateral gene transfer, pathogenicity, environmental adaptations, and interstrain genetic variability. (Liu et al. 2011) A previous report indicated that two highly related prophages SC1 and SC2, which are circular bacteriophage genomes, are associated with 'Ca. L. asiaticus'. The SC1 carries suspected lytic cycle genes and was found in nonintegrated tandem repeats; SC1 genes inhibited bacterial growth consistent with holin and endolysin functions. In contrast, SC2 lacks the lytic cycle genes and replicates as a prophage excision plasmid, encodes putative adhesin and peroxidase genes which are involved in lysogenic conversion (Fleites et al. 2014;Zhang et al.2011).
The prophage lysogenic conversion gene products include extracellular toxins, proteins that alter antigenicity, host effector proteins and enzymes which rescue intracellular survival and proteins that facilitate bacterial attachment to host cells (Boyd and Brüssow 2002) The conventional polymerase chain reaction (PCR) is a DNA ampli cation method used to detect huanglongbing infected plants (Jagoueix et al. 1996); it is inexpensive and easy to perform. 'Candidatus Liberibacter asiaticus' (Las)-speci c primers for conventional PCR are applied by using the Las-speci c primer set Las606/LSS which is a speci c part of the Las 16S ribosomal DNA and is a highly sensitive technique (Fujikawa and Iwanami 2012). The other potential gene locus is the bacteriophage-type DNA polymerase region of the prophage DNA that has played an important role in the evolution of bacterial pathogenicity (Boyd and Brüssow 2002). The terminase prophage gene (representing the corresponding prophage) was indicated to be the SC1-type by the detection of the expected approximately 766 bp DNA amplicon with the speci c primer set 766F/766R based on the sequence of gene CLIBASIA_05610 in the genome of 'Ca. L. asiaticus' strain Psy62 (Liu et al. 2011;Duan et al. 2009). The speci c primer set 817F/817R was used to amplify fragments from the prophage similar to the SC2-type from the Florida strain UF506 of 'Ca. L. asiaticus'. This Florida strain was also shown to carry the SC1-type phage in the nonintegrated tandem repeats, circular form in inoculated periwinkle (Catharanthus roseus) plants (Fu et al. 2015). In this study, population variety among the 'Ca. Liberibacter asiaticus' bacterial pathogen of citrus Huanglongbing disease in Northern Thailand was studied and identi ed by bacteriophages type. In addition, the effect of bacteriophage on symptoms that occured on leaves were also studied.

DNA extraction
The DNA was extracted from leaf midribs by the CTAB method that was modi ed from one previously described by Dellaporta et al (1983). Leaf midrib tissue of citrus (500 mg) was incubated with 4 ml cold grinding buffer for 20 min at 4 °C and then ground nely. The extract was centrifuged at 10,000 g for 5 min at room temperature. The supernatant was transferred to a new tube and centrifuged at 14,000 g for 10 min at room temperature.
The mixture was then centrifuged at 7,000 g for 5 min at room temperature. The aqueous supernatant was transferred to a new tube and combined with an equal volume of isopropanol followed by centrifugation at 14,000 g for 10 min at room temperature. The DNA pellet was washed with 70% ethanol and air dried. The DNA was dissolved in sterile distilled water (15 Ω) and stored at -20 °C.

Effect of Las and bacteriophage on HLB symptoms
The potential role of prophage in disease development leading to variations in HLB symptoms in infected citrus leaves were investigated. Hence, evaluation of the symptoms that occurred on citrus leaves infected by 'Ca. L. asiaticus' induced by non-prophage or various prophages, showed various populations of 'Ca. L. asiaticus' which correlated with symptom that occurred on citrus leaves. The various symptoms were divided and used to assess the severity according to the population of 'Ca. L. asiaticus' with or without prophage.

HLB sample collection
The current research conducted a Huanglongbing (HLB) disease survey in citrus orchards and backyards consisting of 216 samples from 79 location in ve provinces of Northern Thailand which consisted of Chiang Mai 190 samples, Chiang Rai 3, Lampang 11, Nan 5 and Sukhothai 7. Citrus leaf samples were collected from healthy, early and severe symptomatic trees of HLB which exhibited blotchy mottle, yellowing and lamina yellowing on leaves (Fig.1).

PCR ampli cation and sequencing
Detection of 'Ca. L. asiaticus' was based on PCR ampli cation of an expected approximately 500 bp DNA amplicon with primer set Las606/LSS. The prophage terminase gene, SC1-type prophage was indicated by the expected approximately 766 bp DNA amplicon with primer set 766F/766R and SC2-type prophage was indicated by the expected approximately 817 bp DNA amplicon with primer set 817F/817R (Fig.2). The results revealed that 134 of 216 citrus samples from Northern Thailand were infected with 'Ca. L. asiaticus'; the frequency of the pathogen was 62.04%. The breakdown of citrus samples infected with 'Ca. L. asiaticus' were as follows: Chiang Mai province 124 samples, Chiang Rai province 3, Lampang province 3 and Nan province 4. All PCR-positive samples by the primer set Las606/LSS (37 C. reticulata, 1 C. sinensis, 54 C. aurantifolia, 4 C. medica L. var. Medica, 19 C. hystrix and 19 C. maxima) detected the prophage terminase gene of SC1 and SC2-type prophages by 766F/766R and 817F/817R. The SC1 and SC2-type prophage was detected in 37 and 122 samples, respectively. For this result, the population of 'Ca. L. asiaticus' was divided into four groups.The rst group, non-prophage, was found in 10 samples. The frequency was 7.46% (10/134) from the total infected citrus samples. The second group contained SC1-type prophage which was found in 2 samples and the frequency was 1.49% (2/134). The third group containing the SC2-type prophage was found in 87 samples and the frequency was 64.93% (87/134). The fourth group contained both prophages found in 35 samples and the frequency was 26.12% (35/134). The SC2 was found more than the SC1-type prophage because most of the SC2 had the lysogenic cycle as prophage and appeared to lack lytic cycle genes. In contrast, SC1 carried suspected lytic cycle genes as phage, found in this study at only 27.61% (37/134) ( Table 2).

Effect of Las and bacteriophage on HLB symptoms
HLB-infected citrus samples were analyzed and the effect of the prophage terminase gene was compared. The population of 'Ca. L. asiaticus' revealed four groups in Northern Thailand. The rst group was 'Ca. L. asiaticus' which was non-prophage; the frequency was 7.46% (10/134) from the total infected citrus samples; the leaves mainly appeared normal but some leaves had slight yellowing symptoms. The second contained the SC1-type prophage sequence at 1.49% (2/134); the main leaf symptom was a blotchy mottle. The third contained the SC2-type prophage sequence at 64.93% (87/134); the main leaf symptom was interveinal chlorosis. The fourth group contained both prophage sequences at 26.12% (35/134) containing SC1 and SC2-type prophages; the main leaf symptoms were severe blotchy mottle and interveinal chlorosis. Therefore, the citrus leaves were infected with various bacteriophages that affected the symptoms that occurred. These results indicated that the SC1-type prophage caused less of an effect on citrus because of the lytic cycle genes which inhibited bacterial growth. On the other hand, the SC2-type prophage lysogenic cycle stimulated the pathogenicity of 'Ca. L. asiaticus'.
The citrus co-infected with SC1 and SC2-type prophages produced more severe symptoms than the other groups. (Fig. 3)

Discussion
Citrus is one of the most important crops in Northern Thailand including Chiang Mai and Chiang Rai provinces. Epidemics of HLB lead to a signi cant reduction of citrus fruit yield and the abundance of trees. Therefore, this research evaluated the HLB disease and bacteriophages in 'Ca. L. asiaticus' by PCR products ampli ed with speci c primers designed to detect 'Ca. L. asiaticus' and the prophage terminase genes of SC1 and SC2-type prophage. The HLB disease was detected by speci c primers Las606/LSS and ampli cation of the 16S rDNA region of 'Ca. L. asiaticus' which was exactly 500 bp. The PCR results of these 16S rDNA regions con rmed the presence of the 'Ca. L. asiaticus' according to study of Fujikawa asiaticus' and this was the rst report that described the population variation of 'Ca. L. asiaticus' in the two geographical regions between Guangdong and Yunnan provinces, China. The results revealed that the frequency of the prophage terminase gene was 15.8% in Guangdong which has an altitude < 500 m and 97.4% in Yunnan at an altitude > 2,000 m. For our study, we followed this report and used primer set 766F/766R to identify prophage terminase gene (CLIBASIA_05610) which indicated the SC1-type prophage in the 'Ca. L. asiaticus' population. For this study, we followed the report of Liu et al. (2011) and Fu et al. (2015) to detect the prophage terminase genes of SC1(FP1) and SC2 (FP2) of the 'Ca. L. asiaticus' population. These researchers described the variation of a phage DNA polymerase gene, we used two speci c primers 766F/766R and 817F/817R for detection of the presence of phage genome related to the SC1 and SC2 type-prophages in citrus samples collected from Northern Thailand. Our results showed that SC1 and SC2 type-prophages were detected, corresponding with Liu et al. (2011) and Fu et al. (2015). Apart from these two known prophages there could have been other unknown types of prophage or no prophage at all. (Fu et al. 2019). Bacteriophages provide insight into a virulence mechanism of 'Ca. L. asiaticus'. Prophages effected the evolution of bacteria by disruption of the gene in which they integrate, the facilitation of homologous recombination and the expression of new functions directly related to virulence (Fortier and Sekulovic 2013). The citrus samples infected with 'Ca. L. asiaticus' showed several symptoms as follows: asymptomatic, slight yellowing, blotchy mottle and interveinal chlorosis. The symptom was dependant on the number of prophages and the type of prophage. Further evaluation of symptom severity on the citrus leaves revealed that the citrus leaves were infected with various bacteriophages such as SC1 and SC2-type prophage leading to more severe symptoms than 'Ca. L. asiaticus' isolates which have only of SC1 or SC2-type prophages. In addition, HLB-infected citrus sample had less severe symptoms or were non-symptomatic because they lacked prophages. For this study, SC2-type prophages were found in many HLB-infected citrus samples as a result of the lysogenic cycle which promotes intracellular survival and proteins that facilitate bacterial attachment to host cells of 'Ca. L. asiaticus' ( Table 2 Summary of polymerase chain reaction (PCR) detection of 'Ca. L. asiaticus', SC1 and SC2type prophages from various citrus plants